Project Summary/Abstract A critical step in colon cancer development occurs when a cell within a field of Apc- heterozygote mutant cells undergoes Apc loss of heterozygosity (LOH). Understanding the mechanism and cellular consequences of an Apc LOH event could provide important information on how this event might be suppressed to reduce cancer risk. Critical questions that remain unanswered include: Do the events leading to Apc LOH include a failure of cytokinesis? Are all cells that undergo an LOH event at equivalent risk of progression? What genetic and environmental factors can impact Apc LOH? We propose to develop an intestinal organoid system as a malleable experimental platform to study Apc LOH. We will utilize intestinal organoids generated from ESCs since these ?mini-intestines? can be genetically manipulated and grown in the quantities necessary to detect the relatively rare Apc LOH event. We propose to develop this organoid system to study Apc LOH in real-time by linking the ApcMin allele to GFP and the Apc-wild type allele to an RFP reporter and the diphtheria toxin receptor (DTR). We will use these organoids to enumerate and isolate cells that have undergone Apc LOH. We will initially determine how p16 and p53 affect Apc LOH frequency, but envision these organoids being used to assess the impact of a broad range of mutations and polymorphisms. We will study the nature of cells emerging from an Apc LOH event, focusing on their proliferative capacity and their relationship to normal ISCs and cancer stem cells. Finally, we will assess the role of tetraploid cells as precursors for Apc LOH and determine if a novel mitosis-targeting agent that selectively induces apoptosis of tetraploid cells can reduce the frequency of Apc LOH. These exploratory studies are anticipated to establish an experimental system for studying a critical event in colon cancer development; the emergence of Apc-mutant cells in a pre-malignant tissue field. In the long term we envision establishing an integrated experimental pipeline to understand how environmental and genetic factors impact Apc LOH, how cells that have undergone Apc LOH progress to cancer stem cells, and ultimately, how an individual's risk of colon cancer risk can be reduced.